The long-term goal of this project is to develop an in-depth understanding of the structure of Ca2+/calmodulin-dependent protein kinase II (CaMKII) and to relate that structure to the kinase[unreadable]s diverse neuronal functions. CaMKII is the most abundant protein kinase in the mammalian brain and has been ascribed numerous functions including the regulation of neuronal process outgrowth, stabilization of synapse structure, and in the initiation and maintenance of short and long-term synaptic plasticity. Three Aims will be accomplished in the present proposal. The first is to identify binding sites, and the global structural consequence of interactions between CaMKII and substrates as well as other proteins that bind distal to the active site (Aim 1). This goal is motivated by the hypothesis that the oligomeric structure of CaMKII is utilized as a scaffold at synapses on which other proteins can be assembled. The CaMKII-interacting proteins we will examine are localized to synapses, interact with the kinase through unique mechanisms, and include the NMDA receptor, densin-180, and a-actinin. These studies will be accomplished using our successful approach of single particle electron microscopic reconstructions of cryo-preserved specimens. The modular organization of the post-synaptic density will be analyzed using immunogold labeling and 3-D tomographic reconstructions of EM images collected in stain and in cryo (Aim 2). We will specifically examine the distribution of CaMKII and its spatial relationship to PSD- 95, the NMDA receptor, densin-180, a-actinin, protein phosphatase 1 (PP1) and calcineurin (PP2B). We will capitalize on our higher resolution structural information obtained in Aim 1 to help interpret the modular organization of the PSD with a particular focus on CaMKII[unreadable]s potential role as a structural protein (scaffold). Finally, to address CaMKII[unreadable]s role in regulating synaptic morphology, we will determine how CaMKII interacts with the actin cytoskeleton at a structural level and determine what consequences CaMKII interactions have on the biology of actin (Aim 3). We will produce electron microscopic reconstructions of CaMKII bound to actin filaments to establish whether CaMKII can both cross-link actin filaments as bundles and serve as a linker for orthogonal organization of actin filaments. We will also assess if the four mammalian isoforms of CaMKII (a, [unreadable] , ? and d) interact with actin and if they do, what similarities and differences exist between them with regards to polymerization and bundling. We will then determine how the activation status of CaMKII (e.g., Ca2+/CaM-bound or autophosphorylated) impact actin polymerization and bundling. Successful completion of the Specific Aims will add an important understanding CaMKII[unreadable]s role in regulating synaptic function and will provide the data on which to build realistic spatial models of protein-protein interactions at the synapse.